Redescription and phylogenetic position of the enigmatic Neotropical electric fish Iracema caiana Triques ( Gymnotiformes : Rhamphichthyidae ) using x-ray computed tomography

We redescribe Iracema caiana, a monotypic genus of the gymnotiform electric fish family Rhamphichthyidae. Iracema is known only from the type series (holotype and three paratypes) collected from the rio Jauaperi, affluent to the rio Negro in the Amazon basin, and was never collected again. Previous morphological studies were limited to features of external morphology. To study the osteology of Iracema we examined two specimens of different sizes using high-resolution x-ray computed tomography, a non-invasive and non-destructive technique to visualize internal anatomical structures. We describe and illustrate the osteology of Iracema caiana, and present data on morphometrics and external morphology. Contrary to previous hypotheses we propose that Iracema is the sister group to Rhamphichthys based on four synapomorphies: intermuscular bones present in the adductor mandibulae, reticulated texture of opercles, fully ossified Baudelot’s ligaments, and elongate scales above the lateral line in the posterior portion of the body.


Introduction
Iracema caiana Triques is a member of the Rhamphichthyidae, a clade of Gymnotiformes with 15 valid species distributed in three genera (Ferraris, 2003;Lundberg, 2005;Carvalho et al., 2011).Rhamphichthyidae and Hypopomidae constitute the Rhamphichthyoidea, a well supported clade within Gymnotiformes (Mago-Leccia, 1978;Alves-Gomes et al., 1995;Albert, 2001).Iracema Triques was originally based on a unique combination of characters of external morphology.According to Triques (1996a) Iracema can be differentiated from other rhamphichthyids by the presence of one series of irregular to roundish blotches on the sides of the body; a broad and uniformly dark pigmented stripe along the dorsal margin of the body; and an intermediate number of anal-fin rays between Rhamphichthys Muller and Troschel, and Gymnorhamphichthys Ellis.Triques (1996a) briefly discussed putative relationships of Iracema within other rhamphichthyids, hypothesizing it as the sister group to Gymnorhamphichthys based on the shared loss of scales on the anterior portion of the body.
Iracema was first included in a formal phylogenetic analysis by Albert & Campos-da-Paz (1998), who were able to code only 46 characters-states in a data matrix of 170 characters due to absence of osteological information.Most osteological data on Iracema were not available due to a lack of specimens available for clearing and staining; the genus remains known only from four specimens in the type series.To avoid dissection and staining procedures with a limited type series we used an alternative method of visualizing skeletal morphology, high-resolution x-ray computed tomography (HRXCT).HRXCT is a non-destructive technique that allows reconstruction of a virtual skeleton in digital form.Similar approaches to recover osteological data from rare or extinct species have been previously adopted by ichthyologists (Schaefer, 2003;Schaefer & Fernández, 2009;Fink & Humphries, 2010).

Material and Methods
Two specimens were scanned at the High-Resolution Xray Computed Tomography Facility, The University of Texas, Austin.The head and cleithral region of a preserved paratype of Iracema caiana (MZUSP 49205, 345 mm SL) was scanned using the following parameters.FeinFocus microfocal X-ray source operating at 200 kV and 0.16 mA with no X-ray prefilter was employed.An empty container wedge was used.Slice thickness corresponded to two lines in a CCD image intensifier imaging system and equaled 0.0641 mm, with a source-to object distance of 92 mm.For each slice, 1,400 views were taken with three samples per view.The field of image reconstruction was 28 mm, and an image reconstruction offset of 4600 was used with a reconstruction scale of 7000.The dataset consists of 761 1024x1024-pixel HRXCT slices.A second specimen (MZUSP 49205, 235 mm SL) was scanned using the following parameters.An Xradia microXCT scanner operating at 40 kV and 7 W with a 0.15 mm glass X-ray prefilter was employed.An air wedge was used.Source-object distance was 125.3 mm and detector-object distance was 37.1 mm.A total of 521 views were gathered over a rotation of ±104 degrees.The acquisition time per view was 4 seconds.The dataset consists of 909 1024x1024-pixel HRXCT slices, with cubic voxels measuring 43.84 microns.The scan was taken along the long axis of the specimen from the tip of the snout to about the 8-9 vertebrae.Visualizations were produced in the commercial software package VGStudio Max®, at the Ichthyology department of the Academy of Natural Sciences of Philadelphia.Although the renderings appear similar to photographs, they represent the density differences of the biological materials as reflected in their X-ray opacity.Figures were based on still frames captured from digital animations of HRXCT and were prepared using Adobe Photoshop®CS.Homologous bone depicted in figures 3A and 3B were represented by the same spectral colors (hues) in VG Studio Max.These colors differ only in terms of tint (shading density).The shadowing option in VG Studio, used to help visualize objects in 3D produced different effects in these images due to shape differences.Some bones of Iracema caiana are very thin and overlap each other, and therefore it was impossible in the reconstructions to separate them into different color schemes (e.g.preopercle and interopercle).Differing from other fish with published Computed Tomography (CT) scan reconstructions, Iracema has thin and weakly ossified bones (e.g.those from the branchial basket), which are not clearly identified in the CT scans.In addition to the images reconstructed using the software we use the raw X-ray slices data to describe the morphology (e.g.contact between retroarticular and quadrate), however we did not include illustrations, because they have little value compared to the 3d images.The branchial basket was illustrated in the midsagittal plane instead of the usual coronal plane because there is a better resolution and less overlap of bones in these views.
Measurements were made to the nearest 0.1 mm with digital calipers.Standard length (SL), head length (HL) and snout length (SN) follow Nijssen et al. (1976: fig. 1).Standard length as used here is the same measurement as length to end of anal fin (LEA; Mago-Leccia, 1994).Caudal-appendage length (CL) and pectoralfin length (PL) follow Schwassmann (1989: fig.1).Anal-fin length (AL) is the distance from the anterior origin of the anal fin to the posterior end.Body depth (BD) is the greatest depth of the body, which is usually at about half the length of the standard length posterior to end of precaudal vertebrae.Caudal-filament depth (CD) is the depth of caudal appendage at the vertical of the last anal fin ray.Interorbital distance (IO) is the distance between the bony orbits, in the middle part of the orbit.Postorbital length (PO) is measured from the posterior limit of the eyeball to the posterior edge of bony opercle.Distance to posterior nares (PN) is the distance from the tip of snout to the anterior margin of the posterior nares (= measurement Prenasal 2 of Triques, 1996b;fig. 1).Opercular opening (BO) is the distance between the dorsal and the ventral corners of the branchial aperture (measurement 11 of Triques, 1996b;fig. 2).Morphometric data are expressed as percentages of standard length, except subunits of the head, which are expressed as percentages of head length.Osteological terminology is that of Fink & Fink (1981) and Fink & Fink (1996).Cleared and stained specimens (cs) were prepared according to the method of Taylor & Van Dyke (1985).Myological nomenclature follows Winterbottom (1974) and Datovo & Bockmann (2010).Electrocyte rows at the end of anal fin were observed under the transmitted light source in the stereomicroscope.One scale, at about the second third of body (above the lateral line) was removed from MZUSP 49205 (345 mm SL) and stained in alizarin red.Photographs of fish scales were taken using a digital camera attached to an Olympus SZX 12 microscope.Drawings were made using a camera lucida attached to an Olympus SZX 12 microscope.The number of vertebrae to the end of anal fin includes the four of the Weberian apparatus and end with the last vertebra with its hemal spine associated with the last analfin pterygiophore (Lundberg, 2005).Institutional abbreviations are as listed at http://research.calacademy.org/research/ichthyology/catalog/collections.asp.Triques, 1996 Type species.Iracema caiana Triques, 1996a: 91 by original designation and monotypy.

Iracema
Diagnosis.Iracema is distinguished from other rhamphichthyids by the following characteristics: 240-257 analfin rays (vs.139-211 in Gymnorhamphichthys and 304-470 in Rhamphichthys); 15 or 16 pectoral-fin rays (vs.10-13 in Gymnorhamphichthys and 17-22 in Rhamphichthys); a series of dark, rounded pigment blotches on the side of the body along the lateral line (vs.no pigmentation in Gymnorhamphichthys and dark oblique transverse bands or no pigmentation in Rhamphichthys); caudal filament ovoid in cross section, its depth slightly greater than its width (vs.caudal filament laterally compressed in cross section, its depth about three times its width in Gymnorhamphichthys and Rhamphichthys).
Long axis of hyomandibula highly oblique, almost parallel to main axis of neurocranium (Fig. 3).Dorsal (proximal) head of hyomandibula narrow and robustly ossified, with two articulating surfaces.Ventral (distal) limb of hyomandibula narrow and thinly ossified, with slender shelf on its anterior margin at middle of bone.Ventral limb of hyomandibula articulating with metapterygoid anterodorsally, and symplectic anteroventrally (almost certainly by cartilage as in other rhamphichthyoids).Hyomandibula with single large foramen on dorsomedial surface, two foramina on lateral surface, and one on posterodorsal margin, all through which pass cranial nerves.Posterior lateral line nerve with separate lateral foramen located on posterodorsal margin of hyomandibula.Large foramen on lateral surface of dorsal portion of hyomandibula (laterosensory, Trigeminal, and Facial nerve components).Upper jaw consisting of maxilla (m) and premaxilla (p).Maxilla elongate, sickle shape, its dorsal margin concave and ventral margin convex; anterior margin rounded; anterior maxillary process absent; descending process of maxilla narrow, tapering distally.Premaxilla gracile, with small ascending process.Bones of lower jaw consist in dentary, anguloarticular, retroarticular (rar), and coronomeckelian (cm) bones.Dorsal portion of dentary straight; posterior margin deeply forked; anteroventral portion with small hook-shaped process directed posteroventrally.Anguloarticular in close contact with dentary anteriorly and extensively contacting quadrate posteriorly.Anguloarticular condyle socket positioned along posterodorsal margin of lower jaw.Anguloarticular contributing more than retroarticular to joint with quadrate.Retroarticular triangular in shape, anterior margin pointed, dorsoposterior portion contacting quadrate (Fig. 5A).Coronomeckelian bone elongate and teardrop shaped, located at dorsoposterior portion of dentary posterior concavity.Teeth absent in both jaws.
Opercular series.Opercular series consists of preopercle (pop), interopercle (iop), subopercle (sop), and opercle (op; Fig. 6).Preopercle long, anterior margin pointed; without concavity (as in Steatogenys Boulenger and Hypopygus Hoedeman).Dorsal margin concave, most concave in medial portion; anteroventral margin straight, posteroventral margin expanded.Posterodorsal portion of preopercle closely associated with posteroventral margin of hyomandibula.Preopercular-mandibular sensory canal fused with surface of preopercle, with five laterally oriented canal rami leading to pores.Subopercle thin, ventral surface convex and dorsal surface concave.Anterodorsal arm of subopercle long, reaching about same height of dorsoposterior portion of interopercle.Interopercle thin, weakly ossified, triangular in shape, anterior portion in close contact to preopercle.Posterior margin of interopercle convex in contact with subopercle.Opercle broad and fan shaped, dorsal margin concave posteriorly and with small crest anteriorly.Anterior margin of opercle almost straight, surface covered with series of vertical ridges and grooves.Central surface of opercle with reticulated texture.Opercle ventral margin rounded and convex, contacting subopercle anteroventrally.
Branchial basket.Gill arches illustrated in Figure 7.This description includes skeletal support of gills and associated elements, including basihyals (bh), urohyal (uh) and branchiostegals rays (br).Bones of gill arches poorly ossified (or demineralized during preservation), showing some degree of reticulated texture.Dorsal elements of branchial skeleton consisting of four epibranchials (bb), pharyngobranchials (poorly ossified?)and pharyngobranchial toothplate (tp).Epibranchial three with anterior uncinate process and epibranchial four forked posteriorly.Ventral elements of branchial skeleton consisting of five ceratobranchials and three basibranchials.Basihyal fused with basibranchial 1, forming long, rod shaped hollow bone (its core not ossified).Dorsal surface of basihyal rounded, not concave.Basibranchials two and three weakly ossified.Ventral hypohyal (vhh) located anterior to dorsal hypohyal (dhh), these located over anterior ceratohyal (ach).Anterior ceratohyal narrow medially.Posterior ceratohyal (pch) rounded, half ovoid, with distinct notch in dorsal margin.Hypobranchials apparently not ossified.Urohyal fusiform in ventral view; inverted T shape in cross section; with medial ventral slit along long axis, head large with lateral ridges, and anterior portion flat.Posterior portion of urohyal hyperossified forming elongate blade, reaching vertical with third ceratobranchial (cb).Interhyal small and located dorsoposteriorly to posterior ceratohyal.Four branchiostegal rays.First (anterior) branchiostegal ray filamentous, second thin, larger than first, both contacting anterior ceratohyal.Third and fourth branchiostegals lamellar, third contacting limit

Weberian Apparatus. Anterior vertebrae and Weberian
Apparatus illustrated in Figure 8. Neural arches (na) of third and fourth vertebrae and supraneural (sn) meet together forming roof of neural canal.Supraneural adjacent to supraoccipital and neural arch of third vertebra adjacent to exoccipital.Bones of Weberian complex forming socket for dorsal contact between occipital bones of skull and axial skeleton (in Gymnorhamphichthys only supraoccipital articulates with neural complex; Mago-Leccia, 1994: 40).Scaphium (s) barrel-shaped and located anterior to, and slightly ventral to third neural arch.Intercalarium (i) small and round, positioned posterior to scaphium and anterior to tripus (t).Tripus positioned obliquely and descending posteriorly between scaphium and ventral portion of fourth centrum.Tripus narrow anteriorly, broad in median region and tapering posteriorly.First centrum (c1) anteroposteriorly compressed and disk-like shape.Second centrum (c2) larger and with prominent parapophysis behind ossified Baudelot's ligament (bl), extending posterolaterally and fused with fourth parapophysis (pp4) and pleural rib complex.Third centrum located posterior to other components of Weberian complex.Fourth vertebra with fourth parapophysis and pleural rib complex fused to second parapophysis anteriorly.Os suspensorium (os) fused with ventral portion of fourth vertebrae, which extends anteriorly, tapering to reach ventral portion of second centrum.Fifth centrum bearing fifth rib (r5), slightly expanded along antero-posterior axis in midregion.Fifth vertebra bears first neural spine with spinous morphology, located in anterior portion of vertebra, and oriented obliquely posterodorsally.Subsequent ribs widest close to articulation with vertebrae; tapering distally.Anterior chamber of gas bladder not enclosed in bony capsule as in Rhamphichthys (Mago-Leccia, 1994: 40).
Vertebral column.Eighteen precaudal vertebrae (n=2; x-rayed specimens) including vertebrae of Weberian apparatus; 84 vertebrae to end of anal fin (n=1; x-rayed specimen).Neural and hemal spines positioned anteriorly on centrum in anterior vertebrae, transitioning to posterior position on centrum at about vertebra 62. Intermuscular bones (observed in CT scans) composed by dorsal series of myorhabdoi, epicentral bones and series of filamentous bones of anterior epaxial muscles.Epicentral bones in contact with dorsoposterior portion of ribs, rod shaped and somewhat sinusoidal, directed towards posterodorsal portion of body.First two epicentral bones not contacting ribs.Anterior portion of first epicentral bones located slightly anteriorly to vertical with scaphium; second slightly anterior to contact between neural arches three and four, third contacting fourth rib.Dorsal series of myorhabdoi obliquely positioned, anterior and posterior ends branched multiply filamentous bones in anterior dorsal  We were not able to count precisely number of displaced hemal spines, but radiographs show at least more than six in MZUSP 49205, 345 mm SL.Displaced hemal spines all of similar size; absence of enlarged anterior displaced hemal spine (Albert, 2001: fig.35c-d; posteroventral abdominal bone of Hilton et al., 2007: fig. 16b).
Anal fin and supports.Anal-fin rays range from 240-257 rays (n=4).All rays posterior to rays 23-42 branched, no rays branched more than once.Branched portions of rays always adnate from branching origin to distal end of fin.Three analfin pterygiophores between adjacent hemal spines.
Scales.Absence of scales on anterior two-thirds of body.Scales covering entire caudal appendage.Absence of scales in anterior portion of lateral line.Lateral line ossicles embedded in thick layer of skin.Scales above lateral line in posterior portion of body ovoid in shape, elongate on horizontal axis, about twice as long as deep (Fig. 10).

Electric organs.
No information available regarding electric organ discharges (EOD) in Iracema caiana.Hypaxial electric organ extending anteriorly to mental region.Four rows of electrocytes present above posterior end of anal fin.Distribution.Iracema caiana is known only from the type locality on the rio Jauaperi, a tributary of rio Negro in the Amazon basin.Despite a recent survey of several museum collections with large holdings of rhamphichthyid specimens, no additional material of Iracema caiana was identified.This makes Iracema the rarest species of Rhamphichthyidae and one of the rarest of Gymnotiformes (Nogueira et al., 2010).This limited distribution does not seem the result of a sample bias, since the rio Negro basin is relatively well surveyed (Goulding et al., 1988;Chao, 2001).

Discussion
Triques (1996a) diagnosed Iracema caiana from other rhamphichthyids (Rhamphichthys and Gymnorhamphichthys) using a meristic character (anal-fin ray counts) and two color pattern characters.Iracema caiana has 240-257 anal-fin rays, contrasting with 139-211 in Gymnorhamphichthys and 304-470 in Rhamphichthys.Triques (1996a) also noted that Iracema caiana has a color pattern unique within the family, composed of irregularly shaped dark pigment blotches distributed along the sides of the body, and a uniform, broad and darkly pigmented stripe along the dorsal margin of the body.The first of these pigmentation characters is also present in some specimens of Rhamphichthys (an undescribed species; Rhamphichthys sp.

INPA 17646).
Iracema has been consistently considered the sister group of Gymnorhamphichthys, based on derived features of squamation: 1) loss of scales from the anterior two thirds of the body (Triques, 1996a;Albert, 2001: character 16); 2) loss of scales on the anterior middorsum (Albert, 2001: character 15); and (3) possession of tubular lateral line scales (Triques, 1996a; Fig. 3).We observe that all rhamphichthyids have losses of scales to a certain degree.Juveniles Rhamphichthys have no scales on the anterior portion of body, these appearing later during development.In adult Rhamphichthys the squamation of the middorsum is not complete (Albert, 2001: character 16), but rather covered by scattered small round scales.This feature supports the hypothesis that Rhamphichthys is generally hypermorphic in Rhamphichthyidae, with further development in many traits as compared with the other genera.The presence of tubular lateral line scales in the anterior portion of the body is also observed in all juvenile rhamphichthyoids, and also in all adult Rhamphichthys (cf.Triques 1996a: 92) and Gymnorhamphichthys.Albert (2001) diagnosed the clade formed by Iracema and Gymnorhamphichthys by the absence of an ascending process of the endopterygoid (his character 129) and all anal-fin rays unbranched (his character 197).Our examination of computed tomography images of Iracema caiana reveals the presence of an ascending process of the endopterygoid, which is long and very thin (Figs. 3 and 5), a condition shared also by Rhamphichthys but absent and derived in all species of Gymnorhamphichthys.In Iracema caiana, the anal-fin rays posterior to rays 23 to 42 are in fact bifurcated distally, although the rami are either adnate or very close together along most of the length of the fin.This appearance contrasts with the forked appearance of most anal-fin rays along the length of the fin in Rhamphichthys and other rhamphichthyoids.
An alternative sister group relationship between Iracema Intermuscular bones in the adductor mandibulae were noted by Albert (2001: character 44) in Rhamphichthys.We also observe a small number of intermuscular bones in the adductor mandibulae in Gymnotus carapo Linnaeus (USNM 225286), and a similar condition was described for Orthosternarchus tamandua Boulenger (Hilton et al., 2007).According to these authors O. tamandua also possesses intermuscular in the levator operculi and dilator operculi.The presence of intermuscular bones may be correlated with evolution of an elongate snout (although this does not explain their presence in the opercular muscles of Rhamphichthys).We observe intermuscular bones in the levator operculi, dilator operculi, and protractor hyoidei in all species of Rhamphichthys, but not Iracema or Gymnorhamphichthys.
Rhamphichthys and Iracema share a reticulated texture to the central portion of the surface of the opercle, not seen in any other member of Rhamphichthyoidea (Fig. 11).In addition, Rhamphichthys presents a reticulated surface in other bones in the neurocranium, suspensorium and anterior vertebrae, which is not clearly seen in CT reconstructions of Iracema.The presence of reticulated texture to bones (including the opercle) is a derived trait in several other clades of gymnotiforms that inhabit the benthos of large river channels, especially Apteronotidae (except Apteronotus La Cepède s.s.; Albert, 2001: fig. 18: character 82; see also Hilton et al., 2007), and some species of the sternopygid genera Distocyclus Mago-Leccia, Eigenmannia Jordan & Evermann and Rhabdolichops Eigenmann & Allen.The function of the reticulated bone morphology is unknown, but as the pores and pits are usually filled with lipids and the trait may aid in reducing negative buoyancy.
Baudelot's ligament in Gymnotiformes has a single attachment to the neurocranium, separating it distally in two parts where it attaches to the fourth pleural rib and cleithrum (Fink & Fink, 1981).This ligament is partially ossified in some gymnotiforms (e.g.Sternopygus Müller & Troschel; Fink & Fink, 1981: fig. 19d).In Rhamphichthyidae the anterior portion of the ligament is connected to the exoccipital as well as the basioccipital.In the derived condition observed in Rhamphichthys and Iracema, the ligament is totally ossified (Figs. 3 and 8), whereas in Gymnorhamphichthys only the middle portion of the ligament is ossified.
Scales above the lateral line in the posterior portion of body are elongate in the horizontal axis, about twice as long as deep.Elongate scales evolved independently in several gymnotiform clades (Albert, 2001: character 18 andAlbert et al., 2004: fig. 17: character 97).However, within rhamphichthyoids, horizontally elongate scales appear to have evolved only once in a clade formed by Iracema and Rhamphichthys.
The caudal appendage of Iracema caiana differs to a degree from all other rhamphichthyoids and can be considered an autapomorphy.The caudal appendage of Iracema is ovoid in cross section, and at its base (at the posterior end of the anal fin) it is about as wide as it is deep.
The data presented here contradicted all previously published hypotheses of interrelationships of Iracema caiana that regarded it as the sister group to Gymnorhamphichthys (Triques, 1996a;Albert & Campos-da-Paz, 1998;Albert, 2001).These data suggest that Iracema is the sister group to Rhamphichthys and we will not include it in this genus.Also, Rhamphichthys species share several synapomorphies not present in Iracema caiana, e.g. the anterior chamber of gas

, Table 1
345 mm SL; and Triques, 1996a: 92).Anal-fin origin at vertical with pectoral-fin insertion.Absence of thick fold of skin in proximal portion of anal-fin rays.Lateral line complete, with hourglass shape bony tubes.Lateral line with short ventral rami in anterior half of body.Skeletal Anatomy.Skeletal anatomy based on computed tomography (CT) scan data from two specimens of Iracema caiana.

Table 1 .
Morphometrics and meristics of types of Iracema caiana.SD= standard deviation, and N= number of specimens.